Glycoproteins are proteins with covalently linked sugar residues. If these sugar residues are bound to a nitrogen-containing side chain of an amino acid, the sugar is termed "N-linked". If the sugar is bound to a hydroxyl side chain of an amino acid, the sugar is termed "O-linked".
In naturally occurring glycoproteins and in glycopeptide hormones, carbohydrate moieties play key roles in intercellular and intracellular transport of gene products (exit passport hypothesis),.sup.2 as well as extending the biological half-life of the active peptides in vivo (proteolytic protection)..sup.53 Additional roles supported by experimental evidence include the alteration of peptide backbone conformation (protein folding),.sup.3 control of membrane permeability, and molecular recognition (the concept of carbohydrate "antennae")..sup.4 These concepts have been summarized and amplified by Montreuil,.sup.5 and have given birth to the field of "glycobiology"..sup.6 The chemical synthesis of glycopeptides.sup.7 provides an important tool for the study of glycopeptide hormones, glycoproteins and other complex carbohydrate structures found at the cell surface and in the glycocalyx.
Complex glycosides attached to exterior cell surfaces (N-linked glycoproteins, O-linked glycoproteins and glycolipids) are involved in the regulation of cell metabolism, host-pathogen interactions, tumor cell metastasis, cell-cell recognition, and cell adhesion. In order to fully define the roles complex carbohydrates play in these processes, and to thus understand "glycobiology" in its broadest sense, the synthesis of glycopeptides, glycolipids, and their structural analogues is required just as the chemical synthesis of DNA was required to understand molecular biology. O-linked glycopeptides are not as well understood as their N-linked counterparts.
Abnormalities in O-linked glycopeptides are implicated in numerous disease states. Abnormal post-translational modification of the tau protein has been implicated in the formation of neurofibrillary tangles of Alzheimer's disease..sup.8 The antigenic T-epitopes and T.sub.N -epitopes of cell-surface glycopeptides have long been associated with cancer and used as tumor cell markers..sup.9 O-Glycosylated peptide fragments of these two proteins have been synthesized. Insulin-like growth factor (IGF-1),.sup.10 oncofetal fibronectin Val-Thr-His-Pro-Gly-Tyr (SEQ ID NO: 1) fragment,.sup.11 O-glycosyl-somatostatin analogues,.sup.12 O-glycosyltuftsin analogues,.sup.13 O-glycosyl morphiceptin analogues,.sup.14 glycophorin fragments,.sup.15 and mucin fragments.sup.16 have been synthesized for various biological studies.
The synthesis of O-linked glycopeptides is complicated by the acid-lability of glycosides in general and the base-sensitivity (retro-Michael reaction) of the O-serinyl and O-threonyl glycosides in particular..sup.17 Although Boc has been used for N-terminus protection,.sup.12a use of the Fmoc-based peptide coupling strategies (solution.sup. 9b,11 or solid-phase.sup.10 methodology) or Cbz-based strategies.sup.13,14,15 voids acidic conditions for deprotection of the N-termini and appears to be superior. Glycosylation of intact, resin-bound peptides has been attempted,.sup.8 but does not appear to be a generally applicable approach. Acid-labile resin linkers.sup.14,16,18 permit cleavage of the O-linked glycopeptides from the solid-phase support without exposure of the glycoside-bearing residues to strong acids.
The stereoselective synthesis of protected amino acid glycosides remains challenging. Glycosylation of N-acylated .beta.-amino alcohols such as Fmoc-protected or Cbz-protected serine and threonine derivatives, as well as the structurally related ceramides and protected sphingosines.sup.20 is not efficient. Problems encountered by the pioneers.sup.17,21 in this area include low yields and poor .alpha./.beta. selectivity.
Szabo, et al..sup.1 created Schiff base intermediates to facilitate O-linked glycosylation. However, the Szabo, et al. products were protected with methyl groups at the carboxyl terminus of the aminoacid. Methyl groups are not removable under standard peptide synthesis conditions. Thus, these glycosides could not be used to create glycopeptides.
What is needed in the art is an improved method to create amino acid glycosides that are useful to make glycopeptides.